Cell-permeable fluorescent proteins

ABSTRACT

This invention relates to methods and compositions for designing novel fluorescent proteins, preferably to a green fluorescent proteins (GFP). The engineered GFPs are modified by substituting negatively charged amino acids with positively charged amino acids on the exterior of the protein making the protein cell permeable. The ability of the engineered fluorescent proteins to permeate cells obviates the need for transfections, allowing these novel proteins to be used in numerous biological applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending U.S. application Ser. No.11/593,664, filed Nov. 7, 2006, which claims priority to U.S.Provisional Application No. 60/734,210 filed Nov. 7, 2005. Each of theseapplications is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States government support awarded bythe following agencies: NTH GM044783. The United States government hascertain rights in this invention.

BACKGROUND OF THE INVENTION

In biotechnology and genetic research, it is often desirable to usemarker genes or

proteins to test and experiment with genetic processes and geneexpression methodologies. For example, if one seeks to test a genetransfection process, the first gene that is usually employed is a testgene the expression of which can easily be seen in the host. Thus, genesthat encode colorimetric and fluorescent proteins have become populartools for genetic and gene expression research.

One of the popular fluorescent markers that is used in this fashion isthe green

fluorescent protein (GFP). The GFP protein, originally isolated from thejellyfish Aequorea, absorbs light in the visible spectrum and fluorescesin a visible green shade. It has been found that the GFP protein can beexpressed in many different hosts and organisms while still retainingthe characteristic fluorescent activity. One common use of GFP is toplace the GFP gene in tandem with some other gene so that thefluorescence of treated cells will indicate the presence and expressionof transferred DNA.

The GFP protein is a robust and stable molecule. It forms abarrel-shaped tertiary

structure with a series of beta sheets forming a stable cylinder withthe three amino acid chromophore region formed in its interior. Theexterior surface of the barrel shape of the proteins is nativelyanionic, but exhibits a mixture of charged residues to its environment.

Since GFP has been in use as a research tool, many variants of GFP havebeen

developed for a variety of purposes. However, most of the work on theGFP molecule has been on the fluorescent region of the protein to changethe spectral characteristics of the fluorescence or to enhance theamplitude of the fluorescence. The characteristic of cell permeabilityis not usually considered with regard to fluorescent proteins like GFP.Indeed, breaching the plasma membrane barrier is a limiting factor inthe development of proteins and other biomolecules as therapeutics anddiagnostic tools. (See Marafino, B. J., Jr. & Pugsley, M. K. Cardiovasc.Toxicol. 3, 5-12 (2003)). Accordingly, there is much interest indeveloping new means to deliver proteins and other riacromolecules intocells.

BRIEF SUMMARY OF THE INVENTION

The present invention is summarized as relating to an engineeredfluorescent

protein, sui tably green fluorescent protein (GFP) which is modified byreplacing amino acids having negatively charged side chains with aminoacids having positively charged side chains on the exterior of theprotein making the protein cell permeable. The ability of the engineeredfluorescent proteins to permeate cells obviates the need fortransfections, allowing these novel proteins to be used in numerousbiological applications.

In one aspect, the engineered fluorescent protein includes a GFP inwhich at least one residue with negatively charged side chains in theamino acid sequence of a native or an enhanced GFP is substituted with aresidue having positively charged side chains. A suitable substitutionmay occur at any one of amino acid residue positions 17, 19, 21, 111, or124 in the GFP, rendering the protein permeable to cells in culture.

Alternatively, the engineered fluorescent protein includes a GFP inwhich at least three residues with negatively charged side chains in theamino acid sequence of a native or an enhanced green fluorescent proteinare substituted with residues having positively charged side chains.Suitable substitutions occur at amino acid residue positions 17, 19, and21, rendering the protein permeable to cells in culture.

In a related aspect, the engineered fluorescent protein includes a GFPin which at least three residues with negatively charged side chains inthe amino acid sequence of a native or an enhanced GFP are substitutedwith residues having positively charged side chains. Suitablesubstitutions occur at amino acid residue positions 17, 19, 21, 111, and124, rendering the protein permeable to cells in culture.

In another aspect, the engineered form of GFP has at least three aminoacid residues with negatively charged side chains, such as glutamic acidor aspartic acid, in the sequence of the native protein substituted byresidues having positively charged side chains, such as arginine orlysine. The modified GFP is cell permeable (cpGFP).

One example of a cpGFP includes three negative amino acid residuessubstituted with residues having positively charged side chains, such asE17R, D19R, and D21R, substitutions (SEQ ID NO: 6).

Another example of a cpGFP includes five negative amino acid residueswith residues having positively charged side chains, such as E17R, D19R,D21R, E111R, and E124R substitutions (SEQ ID NO: 7).

Another related aspect includes methods for producing the engineeredfluorescent proteins. The method includes culturing host cellscontaining a nucleic acid molecule encoding an engineered GFP underconditions favoring the production of a cpGFP and isolating the cpGFPfrom the host cell.

A related aspect includes an isolated polynucleotide sequence defined bySEQ ID NO:8 encoding the amino acid sequence of the engineered form ofGFP with E17R, D19R, and D21R substitutions. A more suitablepolynucleotide sequence defined by SEQ ID NO: 9includes E17R, D19R,D21R, E111R, and E124R substitutions.

In another aspect the invention includes further modifying the cpGFP toprepare a linker-modified cell permeable fluorescent protein. Thelinker-modified cell permeable fluorescent protein includes (a) apeptide linker region linked at either the amino or carboxyl end of themodified GFP, the linker region susceptible to digestion by a specificprotease; and (b) a fluorescence modifier moiety linked to the end ofthe linker region so as to change the fluorescence characteristics ofthe modified GFP when linked to the protein. The modified protein can beused for cellular assays which are not dependent on GFP expression bythe cell.

The invention is further directed to kits containing any one of theengineered fluorescent proteins described herein or polynucleotidesequences encoding them.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

Other objects, advantages and features of the present invention willbecome apparent from the following specification taken in conjunctionwith the accompanying drawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a representation of the tertiary structure of the native greenfluorescent protein.

FIG. 2 is an illustration showing the location of charged residues onthe exterior of the structure of the green fluorescent protein (GFP).

FIGS. 3A-B show the design of a cationic or cell-permeable variant ofGFP (cpGFP).

FIGS. 4A-B show fluorescence properties of cpGFP.

FIGS. 5A-B show conformational stability of cpGFP.

FIGS. 6A-F show images of the internalization of cpGFP variants intoliving cells.

FIGS. 7A-B show cellular internalization of cpGFP in CHO-K1 and CHO-745cells at high concentration.

FIG. 8 is an illustration of a variant of the present invention showinga cell permeable GFP with a selectively degradable linked region and afluorescence modifying moiety attached thereto.

FIG. 9 is the amino acid sequence alignment for native GFP and itsdifferently colored variants: blue (BFP), yellow (YFP), and cyan (CFP).

FIG. 10 is the DNA sequence for E17R/D19R/D21R/E111R/E124R GFP.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is broadly directed to novel engineered variantsof fluorescent proteins, which are freely permeable to cells. Suitablevariants were created by altering the charge profile of the residues onthe exterior of a fluorescent protein molecule. By making at least oneexterior surface of the protein cationic (positively charged profile),through site-directed mutagenesis, a fluorescent protein capable ofcellular entry was generated. The ability of the engineered fluorescentproteins to permeate cells obviates the need for transfections. Thus,this is the first example of a cell permeable fluorescent protein thatdoes not require an internalization tag. These proteins may be furthermodified for use in monitoring in vivo protease activity and fordeveloping genetic constructs for more diverse biological applications.

To achieve cell permeable fluorescent proteins, as a model, applicantschose to employ the GFP from the jellyfish Aequorea victoria, which hasbeen cloned and the primary amino acid structure has been deduced(Prasher, D. C, et al., Gene 111:229-233 (1992) and FIG. 9 herein).Shown in FIG. 1 is a model for the tertiary structure of native GFP. Theprotein is formed as a stable cylindrical structure, the cylinder beingformed of a series of beta sheets. The chromophore of GFP is positionedinside the cylindrical structure. The chromophore is a hexapeptidecomposed of amino acid residues 64-69 in which the amino acids atpositions 64-67form a heterocyclic ring. The protein is a very stableone, being thermostable over 70° C., but it does not enter into cellswhen placed in medium in which cells are cultured. GFP is awell-characterized acidic protein with a convenient signal for detectingcellular uptake—intrinsic fluorescence. (See, Zacharias, D. A. & Tsien,R. Y. Methods Biochem. Anal. 47, 83-120 (2006)).

Despite these advantages, however, the use of native or wildtype GFP hasa few limitations. One example is the excitation and emission maxima ofwildtype GFP are not within the range of wavelengths of standardfluorescence optics, resulting in low fluorescence intensity. Toovercome this limitation applicants introduced selected point mutationsinto the native GFP sequence. More specifically, applicants madeamino-acid substitutions in enhanced GFP (eGFP), which is the F64L/S65Tvariant and has desirable fluorescence properties. (See, Cormack, B. etal., Gene 173, 33-38 (1996), incorporated by reference here in itsentirety).

The GFP protein, either native (SEQ ID NO: 1) or enhanced (SEQ ID NO:5), was modified to a cationic state, the negatively charged residues onthe protein were substituted with positively charged amino acidresidues. The idea is to make substitutions to convert the chargeprofile of a face or patch of the exterior of the GFP protein from amixed charge to a positive charge. The theory behind this concept is themajority of the saccharides on the exterior of most cells are negativelycharged. Creating a positively charged face or patch on the exterior ofthe GFP protein will attract negatively charged saccharides on the cellexterior resulting in the cationic or cell permeable GFP beingintroduced into the cell.

Shown in FIG. 2 is another view of the exterior of the GFP structurewith one portion of the structure enlarged. In this view, the sidechains of the amino acids of the protein are illustrated. The enlargedportion of the GFP surface is an area of mixed charge side chains in thenative GFP protein. Indicated by the “+” sign are the side chains withpositive charge and indicated with “−” sign are the side chains withnegative charge.

The modifications of the native amino acid sequence exemplified beloware to replace all of the residues with negatively charged side chainsin this region of the protein with alternative residues with positivelycharged side chains. The effect of these substitutions is to make thislarge area of surface of the protein all positively charged, to make aface or region of the surface of the protein positively charged. Therationale being the glycoproteins and glycolipids common on cellsurfaces are predominantly negatively charged, and the electric chargeattraction would bind the modified (cell permeable) GFP to cell surfacesfor eventual ingestion into the interior of the cells. Notably, nocofactors or substrates are required for fluorescence, thus, the proteinmay be used in a wide variety of organisms and cell types.

As used herein the terms “engineered”, “variant”, or “modified” refer toa protein that has been subjected to site-directed mutagenesis, suchthat at least one of the amino acid residues with negatively chargedside chains in the cylindrical body portions (exterior of the protein)are substituted with amino acids having positively charged side chains.Suitable variant proteins include fluorescent proteins, preferably GFP.However, the site-directed mutagenesis approach described herein is notlimited to GFP, but may encompass other colored fluorescent proteinssuch as, cyan, blue, and yellow.

This rational design of a cpGFP proved correct in practice as the cpGFPwas internalized in the presence of cells. Indeed, after introducing themodified cpGFP into the cell culture medium, fluorescencecharacteristics of GFP could then be observed in the cells themselves.However, this same phenomenon did not occur in other cell lines whichwere deficient in glycoaminoglycans (GAGs), the negatively chargedchains on the exterior of the cell surfaces.

Thus, broadly characterized, one embodiment of the invention is anengineered fluorescent protein, preferably GFP, having amino acids withnegatively charged side chains substituted with amino acids havingpositively charged side chains on the exterior of the protein, renderingthe protein cell permeable. It is noted that such substitutions can bemade to the native GFP, enhanced GFP as described herein or GFP havingcombinations of other mutations which would not interfere with thepositive charge profile of an engineered cpGFP.

More specifically, in another embodiment, the invention provides anengineered form of a fluorescent protein, preferably GFP, that has atleast one native amino acid residue with a negatively charged side chainsubstituted by an amino acid residue having a positively charged sidechains. Locations of amino acid substitutions include positions 17, 19,21, 111, and 124. The modified GFP is cell permeable.

The substitutions to the enhanced GFP sequence described in the examplebelow are substitutions of glutamic acid (E) and aspartic acid (D)substituted in each instance with an arginine (R) residue. Arginine hasa positively charged side chain as opposed to the negative charges onglutamic acid and aspartic residues. The substitutions are indicated inthe specification here by the common nomenclature such as E17R, whichindicates that the glutamic acid residue normally present at residue 17in the amino acid sequence is substituted by an arginine residue at thatlocation. The other possible substitutions would include otherpositively charged amino acids, such as lysine, for these or othernegatively charged residues.

As used herein the phrase “positively charged amino acids” includesnaturally occurring or non-naturally occurring basic amino acids.Preferred amino acid residues include but are not limited to arginine(R), lysine (K) and histidine (H).

As used herein the phrase “negatively charged amino acids” includesnaturally or non-naturally occurring acidic amino acids. Preferred aminoacid residues include, but are not limited to glutamic acid (E) andaspartic acid (D).

Another embodiment of the invention includes an engineered fluorescentprotein, preferably GFP, wherein the protein has E17R, D19R, D21R,E111R, and E124R substitutions. It should be noted these five amino acidsubstitutions described in the example below are sufficient to achievecell permeability. It is contemplated that more such substitutions arepossible to increase the permeability of the modified GFP to cells.These five substitutions are all in the cylindrical body portions of theGFP tertiary structure. Since this change to the protein sequence occursfar from the region of the chromophore of the protein, the change doesnot affect the fluorescent properties of the molecule in any way. Thus,these same substitutions will similarly affect on other modified formsof GFP where the modifications to the other forms are intended to affectthe fluorescence characteristics of the protein.

In addition to conferring cell permeability to a native or an enhancedfluorescent proteins as described herein, it is contemplated that onecould confer cell permeability to other modified proteins, preferablyGFP, having a combination of mutations resulting in desirableproperties. The properties could vary from higher expression inmammalian cells, higher fluorescence intensity under UV or white lightillumination, etc. A number of other genetic modifications could be madeto GFP resulting in variants for which spectral shifts correspond tochanges in the cellular environment such as pH, ion flux, and thephosphorylation state of the cell which can be accomplished usingstandard techniques routine in the art. Thus, in addition to possessingsuch properties, the fluorescent protein may be endowed with a cationic“patch” (by making the exterior surface charge positive) giving theprotein affinity for negatively charged GAGs on the cell surface.

Another embodiment includes methods for making the engineered GFPs. Themethod includes culturing host cells containing a nucleic acid molecule(an expression vector) encoding an engineered GFP alone or in tandemwith other genes under conditions favoring the production of a cpGFP andisolating the cpGFP from the host cell. These engineered proteins can beproduced using both eukaryotic and prokaryotic cells.

Unless otherwise specified, a nucleotide sequence encoding an amino acidsequence includes all nucleotide sequences that are degenerate versionsof each other and that encode the same amino acid sequence.

In conjunction with production of the engineered proteins, specificplasmids, expression vectors, promoters, selection methods and hostcells are disclosed and used herein and in the Examples, otherpromoters, vectors, selection methods and host cells, both prokaryoticand eukaryotic, are well-known to one of ordinary skill in the art andmay be used to practice the present invention without departing from thescope of the invention or any of the embodiments thereof.

The cpGFP and modifications thereof will have utility as fluorescenttags and fluorescent enzyme substrates in living cells. For example, inthe examples below, a small-molecule fluorescent quencher is attached tothe cpGFP via a peptide tag that is cleavable by HIV protease. Thismolecule could be used to (1) screen for viruses, such as HIV in humancells and (2) separate infected cells which would have unquenched GFPfluorescence from uninfected cells with quenched GFP fluorescence.

Another embodiment of the invention includes a linker-modified cellpermeable fluorescent protein employed to assay the presence of proteasein cells. The linker-modified protein including (a) a modified GFP inwhich at least three of the negatively charged amino acid residues,preferably glutamic acid or aspartic acid, in the native or enhanced GFPare substituted by residues having positively charged side chainsincluding arginine or lysine residues, the modified GFP being permeable;(b) a peptide linker region linked at either the amino or carboxyl endof the modified green fluorescent protein, the linker region susceptibleto digestion by a specific protease; and (c) a fluorescence modifiermoiety linked to the end of the linker region, the fluorescence modifierchanging the fluorescence characteristics of the modified greenfluorescent protein if linked to the protein, so the presence of theprotease can be assayed in cells by placing the linker-modified cellpermeable fluorescent protein in the presence of the cells. Botheukaryotic and prokaryotic cells are detectable using the engineeredcell permeable fluorescent proteins. Suitably, host cell lines mayinclude but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, Jurkat,HEK-293, and W138.

As used herein the term “linker region” refers to a peptide moiety. Thepreferred linker moiety is a peptide between about one and 30 amino acidresidues in length, preferably between about two and 15 amino acidresidues. In one example, the linker has the amino acid sequenceTSFNFPQITC (SEQ ID NO: 13). The linker region is designed to bedigestible by a specific protease, such as HIV-1 protease. Other peptidemoieties recognized in the literature are within the scope of theinvention.

Attached covalently to the carboxyl terminal of the linker is a“fluorescence modifier moiety”. This moiety, suitably TMR (tetramethylrhodamine) has the effect of modifying the fluorescence characteristicsof GFP and other fluorescent proteins (YFP, BFP, CFP), if and only ifthe modifier is in close proximity to the GFP itself. If the TMR is inclose proximity to the GFP, the fluorescence of the GFP is higherrelative to the normal GFP where the TMR is in further proximity. Otherfluorescence modifier moieties which may interact with GFP are alsoencompassed herein, such as, eosin, eosin Y or eosin B.

A related embodiment includes a method for assaying cells in culture foractivity of specific protease, suitably HIV-1 protease. The methodincludes placing the linker-modified cell permeable GFP in culture withcells and then measuring the fluorescent characteristics of the cells,wherein a higher than normal fluorescence is indicative of proteasepresent in the cells.

The materials of the invention are ideally suited for a kit tofacilitate a variety of applications. In preferred embodiments thecompositions (polynucleotides and polypeptides) of the invention may beassembled into kits for use in labeling target polypeptides with thepresent cpGFPs or in vivo monitoring of protease activity in a cellsample.

The subject kits generally include at least one container containing,for example, a cpGFP or ε cpGFP having a peptide linker region and afluorescence modifier moiety linked thereto as described herein (i.e.,linker-modified cell permeable GFP). Preferably the kit may include anyone of the engineered cpGFP proteins (SEQ ID NOs: 6 and 7) orpolynucleotide sequences encoding these proteins (SEQ ID NOs: 8 and 9).In the subject kits, the above components may be combined into a singleaqueous composition or separate as different or disparate compositions,e.g. in separate containers. Other reagents may be included in the kit.

In addition to the above components, the subject kits may furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the kits in a variety of forms includingbut not limited to paper or computer readable form.

The following examples are provided as further non-limitingillustrations of compositions and methods for practicing the claimedembodiments.

EXAMPLES Materials

The Plasmid pRSET_(B), containing a cDNA for enhanced GFP (eGFP) whichdirects the production of a DNA for eGFP was obtained from Dr. S. JamesRemington (University of Oregon). Since the modifications to eGFP fromthe native sequence were to enhance fluorescence, the use of GFP ratherthan native GFP was considered equivalent. Oligonucleotides forsite-directed mutagenesis of this DNA (eGFP cDNA) were obtained fromIntegrated DNA Technologies (Coralville, Iowa) and had the sequences: 5′CACTGGAGTTGTCCCAATTCTTGTTCGTTTACGTGGTCGTGTTAATGGGCACAAATTT TCTGTCAGTGG3′ (SEQ ID NO: 10 and its reverse complement; sites of the E17R, D19R,and D21R substitutions are underlined), 5′

CGGGAACTACAAGACACGTGCTCGTGTCAAGTTTGAAGGTGATACCC 3′ (SEQ ID NO: 11 andits reverse complement; the site of the E111R substitution isunderlined), and 5′ CCCTTGTTAATAGAATCCGTTTAAAAGGTATTGATTTTAAAG 3′ (SEQID NO: 12 and its reverse complement; the site of the E124R substitutionis underlined). DH5a and BL21(DE3) competent Escherichia coli cells werefrom Stratagene (La Jolla, Calif.).

Example 1 Site-Directed Mutagenesis

A variety of site-directed mutagenic techniques may be used to preparethe GFP mutants described here. Such methods are well known and standardin the literature. To practice the invention, plasmids that direct theproduction of eGFP variants were obtained from plasmid pRSETB by usingthe QuikChange mutagenesis kit (Stratagene, La Jolla, Calif.) withcomplementary oligonucleotides described above. Three successive roundsof mutagenesis yielded DNA encoding eGFP with five substitutions: E17R,D19R, D21R, E111R, and E124R. The engineered protein with at least threeof these changes (i.e., E17R, D19R, and D21R) and preferably all five ofthese changes is referred to as “cationic GFP” or simply “cpGFP”. Theamino acid sequences of GFP and several of its differently coloredvariants are set forth in FIG. 9 attached below, which also shows theacidic residues mutated to arginine in the modified GFP disclosed above.FIG. 10 sets forth the nucleotide sequence of a coding region sufficientto express E71R,D19R,D21R,E111R/E124R cell permeable GFP (SEQ ID NO: 9).

Example 2 Protein Production

Plasmids containing the sequences for and directing the production ofeGFP and its variants were transformed into BL21(DE3) cells and colonieswere selected on Luria-Bertani (LB) agar plates by their resistance toampicillin. Starter cultures (25 mL) of LB medium containing ampicillin(200 μg/mL) were inoculated with a single colony of E. coli and grown at37° C. with shaking at 200 rpm to an optical density of 0.6 at 600 nm.Cultures (1.0 L) of the same medium were inoculated with 4 mL of thestarter culture and grown at 37° C. with shaking at 300 rpm to anoptical density of 0.6 at 600 nm. Cultures were then cooled to 15° C.,and gene expression was induced by the addition of isopropylβ-D-1-thiogalactopyranoside (IPTG) (final concentration: 1 mM). Cultureswere grown at 15° C. with shaking at 300 rpm for 18 hours, and cellswere harvested by centrifugation (5,000 rpm for 10 min) in a BeckmanCoulter Avant J-20 XPI centrifuge using a JLA 8.1 rotor. Cell pelletswere either frozen or used immediately to purify protein.

Example 3 Protein Purification

Cell pellets were resuspended in 10 mL of ice-cold cell lysis buffer,which was 50 mM sodium phosphate buffer (pH 7.2) containing 500 mM NaCl(500 nM) and PMSF (1 mM). Cells were lysed by sonication (50% duty/50%output) five times for 30 seconds. Cell debris was removed bycentrifugation at 22,000×g for 60 minutes at 4° C. in a Beckman OptimaXL-80K ultracentrifuge using a 60 Ti rotor. Clarified cell lysate wasdialyzed for at least 2 hours against PBS+, which was 50 mM sodiumphosphate buffer (pH 7.2) containing NaCl (636 mM) before loading onto acolumn of Ni-NTA agarose resin (Qiagen, Germany). The resin was washedwith the same buffer containing 20 mM imidazole before eluting with 50mM sodium phosphate buffer (pH 7.2) containing NaCl (636 mM) andimidazole (500 mM). The fractions containing green-colored protein (thatis, eGFP) were pooled and diluted 1/10 with water to lower the saltconcentration. The eGFP was then loaded onto a 5-mL column of HiTrap SPFF sepharose resin (Amersham Biosciences, Piscataway, N.J.). Protein waseluted with a 100-mL linear gradient (50 mL+50 mL) of NaCl (0-1.00 M) in50 mM sodium phosphate buffer (pH 7.2).

Fractions containing green-colored protein were pooled and dialyzedagainst 50 mM sodium phosphate buffer (pH 7.5) containing NaCl (636 mM).The N-terminal histidine tag was removed as described previously (Hansonet al. J. Bio. Chem., 279, 13044-13053 (2004)). Briefly, protein wasincubated with α-chymotrypsin (1:50 w/w) for 20 hours at roomtemperature. Chymotrypsin degrades the N-terminal tag but does notcleave the GFP protein (Hanson et al. 2004). Protein was concentratedwith Vivascience 5000 MW spin columns, and protein concentration wasdetermined by optical absorbance at 280 nm (ε₂₈₀=19890 M⁻¹cm⁻¹) or bythe BioRad protein assay.

Example 4 Cell Internalization

Chinese hamster ovary cells (CHO-K1), glycosaminoglycan-deficient celllines (CHO-677 and CHO-745) and HeLa cells were obtained from theAmerican Type Culture Collection (ATCC) and maintained according torecommended instructions. The day before incubation with a protein,cells were seeded onto 4- or 8-well Lab-Tek II Chambered Coverglasstissue culture dishes (Nalge Nunc International, Naperville, Ill.) toyield 75% confluency the next day. The following day, protein solutions(in PBS + 500 mM NaCl) were added to cells in 200 μL (volume of theprotein solution was < 1/20 of the total volume) of medium or PBScontaining magnesium (1 mM) and calcium (1 mM). Protein was incubatedwith cells for the indicated time. The cells were then washed threetimes with PBS containing magnesium and calcium prior to visualization.In some experiments, cell nuclei were counterstained with Hoescht 33342for 5 minutes prior to washing with PBS. Internalization was visualizedon a Nikon CI laser scanning confocal microscope equipped with 60× and100× lenses. The cpFGP is capable of crossing cell membranes rapidly andcan be visualized by fluorescence microscopy in the cytoplasm ofcultured human cells in under one hour.

Example 5 Conformational Stability

The conformational stability of GFP variants was determined by followingthe change in fluorescence as a function of denaturant concentration(Stepanenko, O. V. et al. Biochemistry 43, 14913-14923 (2004)). GFPproteins (1-5 nM) were incubated in 96-well flat-bottom plates (totalvolume: 100 μL) in 50 mM sodium phosphate buffer, pH 7.5, containingNaCl (500 mM) and guanidine-hydrochloride (Gdn-HCl) (0-6.30 M) for 24 hat room temperature. Fluorescence intensity was determined using a TecanUltra 384 fluorescence plate reader. Data were fitted to a two-stateunfolding mechanism and could be used to calculate the standard freeenergy of denaturation: delta G° (=−RTlnK), where R is the gas constant,T is the absolute temperature, and K is the equilibrium constantcalculated from the experimental data with the equation (see Tanford, C.Adv. Protein Chem. 23, 121-282 (1968): K=[y_(N)−y]/[y−y_(D)]. The valueof y is the observed fluorescence value, and y_(N) and y_(D) are the yvalues for the native and denatured states, respectively.

Example 6 Fluorescent Properties

Fluorescence measurements were made with a QuantaMaster 1 photoncounting fluorometer equipped with sample stirring (Photon TechnologyInternational, South Brunswick, N.J.). Fluorescence excitation andemission spectra were obtained in PBS+ buffer using a 2-nm slit widthand 1-nm/second scanning rate. Quantum yield (Φ) was determinedfollowing a method described previously (Cubitt et al. Methods CellBiol, 58, 19-30(1999)). Briefly, the ultraviolet absorbance at 490 nm ofeGFP in PBS+ was matched to that of a fluorescein standard in 0.1 N NaOHof known quantum yield (Φ_(f)=0.95 in 0.1 N NaOH). The area under theemission spectra from 500-700 nm (excitation at 490 nm) was determined,and the ratio (eGFP area/fluorescein area) was used to calculate thequantum yield of eGFP (eGFP area/fluorescein area=Φ cGFP/Φ fluorescein).

Example 7 Electrostatic Potential Diagrams

Electrostatic potential diagrams were made by using the atomiccoordinates for F64L/S65T/Y66L GFP (Protein Data Bank entry 1S6Z4, seeRosenow, M. A., Biochemistry 43, 4464-4472 (2004)) and the programMacPyMol (DeLano Scientific, South San Francisco, Calif.). Defaultsettings were used except that the Coulomb dielectric was set to be 80.A model of cpGFP was created and likewise modeled by using the programMacPyMol.

Example 8 Preparation of Linker-Modified Cell Permeable FluorescentProteins

The Plasmid pRSETB, containing a cDNA for enhanced GFP (eGFP) whichdirects the production of a DNA for eGFP was obtained. As indicatedabove, since the modifications to eGFP from the native sequence were toenhance fluorescence, the use of GFP rather than native GFP wasconsidered equivalent. Oligonucleotides for site-directed mutagenesis ofthis DNA (eGFP cDNA) were obtained from Integrated DNA Technologies(Coralville, IA) and had the sequences: 5′ GGCATGGATGAACTATACAAAACGGTGTCGTTCAATTTCCCGCAGATCACGTGTTAATAAGGATCCGAGCTCGAGATCTG 3′ (SEQ ID NO:14) and reverse complement where the HIV-PR cleavage site is underlined(corresponding to TVSFNFPQITC (SEQ ID NO: 15)). The primers to deleteresidues 230-238 of eGFP had the following sequence: 5′

GAGTTTGTAACAGCTGCTGGGATTACGGTGTCGTTCAATTTCCCG 3′ (SEQ ID NO: 16) andreverse complement. DH5α and BL21(DE3) competent Escherichia coli cellswere from Stratagene (La Jolla, Calif.). The primers to make themutations C48S and C70V respectively were 5′CCAATTGCTACCAGAGCAAGTCCACCATGAGAATCACCG 3′ (SEQ ID NO: 17) and 5′CTCTCACTTATGGTGTTCAAGTCTTTTCAAGATACCCAG 3′ (SEQ ID NO: 18) withrespective complements. DH5α and BL21(DE3) competent cells were fromStratagene (La Jolla, Calif.).

One potential application for cpGFPs is as cell permeable substrates forcellular enzymes. In this case, cpGFP would be fused to anotherfluorescent molecule, such as a fluorophore or another fluorescentprotein, through an amino acid linker. To prepare the linker-modifiedGFP variant described here, plasmids that direct the production of eGFPvariants were obtained from plasmid pRSETB by using the QuikChangemutagenesis kit (Stratagene, La Jolla, Calif.) with complementaryoligonucleotides described above. These oligonucleotides encode for alinker sequence that corresponds to a naturally occurring cleavage sitefor HIV-1 protease. However, this amino acid linker can be replaced withany amino acid sequence of length 2 to 20 amino acids that may or maynot encode for a variety of other protein recognition sequences. In theexample where cpGFP would be fused to a small molecule, this linkerwould also contain the amino acid cysteine, that could react withthiol-reactive small molecules. In this case, it may be necessary tomake substitutions to the naturally occurring cysteine residues withinGFP at positions 48 and 70 to ensure site-specific modification of thecysteine in the aforementioned linker sequence. The native cysteineresidues in GFP at positions 48 and 70 may be replaced with serine andvaline using site-directed mutagenesis and the primers listed above.These substitutions do not interfere with the fluorescent or cellpermeable properties of cpGFP.

To produce the linker-modified GFP mutants, plasmids containing thesequences for and directing the production of eGFP and its variants weretransformed into BL21(DE3) cells. Colonies were selected and the cellswere cultured as described above. The IPTG-induced cells were harvestedand the cell pellets were either frozen or used immediately to purifythe engineered protein. To purify the linker-modified cpGFP proteinscell pellets were subjected to the same purification protocol asdescribed above for other engineered fluorescent proteins. The purifiedlinker-modified cell permeable fluorescent proteins may be used for avariety of in vitro and in vivo applications.

Discussion

GFP is an acidic protein, having a net charge (i.e., Arg+Lys−Asp−Glu) ofZ=−9 at neutral pH. Applicants noted that one face of GFP is variegatedwith acidic and basic residues (FIG. 3A). Shown in FIG. 3A is a ribbonmodel depicting the location of the five anionic residues in GFP thatwere replaced with arginine to yield a surface comprised often cationicresidues. The fluorophore is depicted in space-filling mode. Applicantschose to replace the five acidic residues (Glu17, Asp19, Asp21, Glu111,and Glu124) on this face with arginine. These acidic residues reside onthree, adjacent Beta-strands, proximal to five basic residues (Lys107,Arg109, Lys113, Lys122, and Lys126). Accordingly, these fivesubstitutions created a highly cationic patch on the surface of eGFPyielding a nearly neutral (Z=+1) variant that applicants refer to ascell-permeable GFP (cpGFP) seen FIG. 3 b). FIG. 3B shows a space-fillingmodel depicting the effect of the arginine substitutions on theelectropotential surface (blue: cationic; red: anionic).

Applicants produced cpGFP in Escherichia coli. (See, Hanson, G. T. etal. Biochemistry 41, 15477-15488 (2002)). Cation-exchange chromatographywas especially efficacious in the purification of cpGFP, affordingnearly homogeneous protein. The fluorescence properties of cpGFP werefound to be nearly identical to those of eGFP (see FIG. 4).Specifically, FIGS. 4A-B show fluorescence properties of cpGFP. (a)Fluorescence excitation ( - - - ) and emission (—) spectra for cpGFP(blue) and eGFP (green). Data were collected in 1-nm increments with ascan rate of 5 nm/s. (b) Raw data for determination of the quantum yield(Φ) for cpGFP. Solutions of cpGFP (blue) and fluorescein (green) (Φfluorescein=0.95 in 0.10 N NaOH) of equal absorbance at 490 nm werediluted in PBS+ (for cpGFP) or 0.10 N NaOH (for fluorescein), and thearea under the emission spectrum curves from 500-700 nm was determined.The quantum yield of cpGFP was determined with the equation: Φ cpGFP=Φfluorescein(cpGFP area/fluorescein area).

Formation of the GFP fluorophore requires its proper folding. (See Cody,C. et al., Biochemistry 32, 1212-1218 (1993); and Waldo, G. et al., Nat.Biotechnol. 17, 691-695 (1999)). Moreover, use of GFP requires theretention of its conformational stability in biological assays.Replacing anionic residues with cationic ones can alter proteinstability, though this effect is not readily predictable. (See, Pace, C.et al., Protein Sci. 9, 1395-1398 (2000)). Hence, applicants usedchemical denaturation to ascertain the effect of site-specificcationization on the stability of eGFP. Applicants observed that bothcpGFP and eGFP have unfolding midpoints at C_(1/2)=(3.1±0.3) Mguanidine-HCl (see FIG. 5). Specifically, FIGS. 5A-B show conformationalstability of cpGFP. The unfolding of cpGFP (blue) and eGFP (green) wereinduced by Gdn-HCl. (a) Dependence of normalized fluorescence intensityon denaturant concentration. The midpoint of the transformation wasfitted to a sigmoidal dose-response curve with the program Prism(Graphpad Software, San Diego, Calif.). The midpoint of the transitioncorresponds to the value of C_(1/2), which is the concentration ofdenaturant at which the protein is 50% unfolded at equilibrium, (b)Dependence of ΔG° on Gdn-HCl concentration. Data were fitted to abilinear regression such that the slope (m) is the dependence of ΔG° ondenaturant and the y-intercept approximates the free energy of theprotein in the absence of denaturant (ΔG°˜ΔG(H2O)). Thus, the creationof a cationic patch did not have a deleterious effect on conformationalstability.

Cellular internalization of GFP can be visualized by fluorescencemicroscopy. Applicants incubated cells with increasing concentrations ofeither cpGFP or eGFP for known times at 37° C. Prior to visualization,cells were placed in fresh medium for 1 h to allow for theinternalization of any protein bound to the cell surface. Specifically,HeLa cells were incubated with cpGFP (a, 10 μM; b, 1 μM; c, 0.1 μM) andeGFP (d, 10 μM) for 3 h in Opti-MEM medium at 37° C. Cells were thenplaced in fresh medium for 1 h and stained with Hoescht 3342 (blue) andpropidium iodide (red) for 15 min prior to visualization by confocalmicroscopy. Fluorescence intensity within living cells wasdose-dependent, increasing at high concentration of cpGFP (FIG. 6 a-c).Although a small amount was detectable in the cytosol, cpGFP wasobserved primarily in vesicles. This localization is similar to thatobserved with cationic peptides, such as polyarginine. (See, Fuchs, S.M. & Raines, R. T. Biochemistry 43, 2438-2444 (2004)). Insignificaitfluorescence intensity was observed in cells incubated with eGFP (FIG. 6d).

Glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitinsulfate (CS) on the cell surface can mediate the binding of cationicpeptides and proteins. (See, Fuchs, S. M. & Raines, R. T. Cell. Mol.Life Sci. 76, 1819-1822 (2006); Fuchs, S. M. & Raines, R. T.Biochemistry 43, 2438-2444 (2004); and Richard, J. P. et al. J. Biol.Chem. 280, 15300-15306 (2005)). To probe for a role for GAGs in cpGFPinternalization, applicants compared cell-surface binding ar.d cellularinternalization of cpGFP in wild-type CHO-K1 cells to that in a CHO cellline that is deficient in GAG biosynthesis. Specifically, CHO-K1 andCHO-745 cells (which are GAG-deficient) were incubated with cpGFP (2 μM)for 3 h at 37° C. in Opti-MEM medium. Cells were then placed in freshmedium for 1 h and stained with Hoescht 33342 (blue) and propidiumiodide (red) for 15 min prior to visualization.

In wild-type CHO-K1 cells, cpGFP was observed to bind to the cellsurface and undergo internalization (FIG. 6 e). In CHO-745 cells (whichare deficient in HS and CS), there is little internalization of cpGFP(FIG. 6 f). At a 10-fold higher protein concentration, cpGFP isinternalized in the GAG-deficient cell line (see FIG. 7). Specifically,FIGS. 7A-B show cellular internalization of cpGFP in CHO-K1 and CHO-745cells at high concentration. cpGFP (20 μM) was added to Opti-MEM mediumcontaining CHO-K1 (a) and CHO-745 (b) cells, and incubated for 3 h at37° C. Cells were then placed in fresh medium for 1 h, and stained withHoescht 33342 (blue) prior to visualization. Scale bars: 50 μm. Similarresults were obtained with another GAG-deficient cell line, CHO-677(data not shown). Apparently, cpGFP internalization relies largely, butnot exclusively, on the interaction with cell surface GAGs.

GFP and its variants are in widespread use in cell biology. (See,Zacharias, D. A. & Tsien, R. Y. Methods Biochem. Anal. 47, 83-120(2006); and Ward, T. & Lippincott-Schwartz, J. Methods Biochem. Anal.47, 305-337 (2006)). Among these variants, cpGFP is unique in obviatinga need for transfection to infuse mammalian cells with a fluorescentprotein, and thus could have numerous applications, both in vitro and invivo. (See, Hoffman, R. M. Nat. Rev. Cancer 5, 796-806 (2005)). Moregenerally, our data demonstrate that an exogenous chain of arginineresidues (which is readily susceptible to proteolysis) is not anecessary component of a cell-permeable protein. (See, Fuchs, S. M. &Raines, R. T. Protein Sci. 14, 1538-1544 (2005)). Applicants anticipatethat site specific cationization will be a useful means to endowproteins other than GFP with cell permeability.

Furthermore, a unique application of the cell permeable GFP isillustrated in FIG. 8. FIG. 8 shows a linker-modified cell permeableGFP. This embodiment of the engineered GFP has attached to it a linkerregion, specifically a peptide as set forth in sequence TSFNFPQITC (SEQID NO: 13). This peptide linker region is designed to be digestible by aspecific protease, in this case the HIV-1 protease. Attached covalentlyto the carboxyl terminal of the linker is a molecule designed as TMR(tetramethyl rhodamine) which has the effect of modifying thefluorescence characteristics of GFP if and only if the modifier is inclose proximity to the GFP itself. If the TMR is in close proximity tothe GFP, the fluorescence of the GFP is at 575 nm, as opposed to thenormal fluorescence at 515 nm characteristic of enhanced GFP. In thisusage, close proximity means close enough to alter the fluorescencecharacteristics of the GFP.

The linker-modified cell permeable fluorescent protein can be introducedinto the medium in which cells are cultured, resulting in the proteinentering the cell. If the cells into which the GFP permeated containHTV-1 protease, the protease will digest the linker and the GFP in thosecells will fluoresce at 515 nm in those cells. If the cells contain noHTV-1 protease, the linker will not be digested and the GFP in thosecells will fluoresce at 575 nm. Thus a simple fluorescent assay forexpression of HIV-1 protease is enabled. This assay can be used to testor sort individual cells or can be used to test or assay a cell cultureor cell sample from a host. This same methodology may be used to providean assay for the expression of any protease of interest in any cell typeor cell source.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is understood that certain adaptations of theinvention are a matter of routine optimization for those skilled in theart, and can be implemented without departing from the spirit of theinvention, or the scope of the appended claims.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. It is understood, however, that examples and embodimentsof the present invention set forth above are illustrative and notintended to confine the invention. The invention embraces all modifiedforms of the examples and embodiments as come within the scope of thefollowing claims.

1.-9. (canceled)
 10. An engineered fluorescein protein comprising: a) afluorescent protein differing in amino acid sequence from the referenceprotein of (i) SEQ ID NO: 1-3 or 5, wherein the difference consists ofsubstituting a glutamic acid or a aspartic acid with an arginine or alysine at positions 17, 19, and 21 of SEQ ID NO: 1-3 or 5, or (ii) SEQID NO: 4, wherein the difference consists of substituting a glutamicacid or an aspartic acid with an arginine or a lysine at positions 18,20, and 22 of SEQ ID NO: 4, where the modified protein is permeable to acell; b) a peptide linker region linked at either the amino or carboxylend of the modified fluorescent protein, the linker region susceptibleto digestion by a specific protease; and c) a fluorescence modifiermoiety linked to the end of the linker region, the fluorescence modifierchanging the fluorescence properties of the modified fluorescent proteinto which it is linked.
 11. The engineered protein as claimed in claim 10wherein the linker has the amino acid sequence TSFNFPQITC (SEP ID NO:13) and the protease is HIV-1.
 12. An engineered protein as claimed inclaim 10, wherein the fluorescence modifying moiety is a tetramethylrhodamine.
 13. An engineered protein for use in detecting proteaseactivity in a cells, the engineered protein comprising: a fluorescentprotein differing in amino acid sequence from the reference protein of(i) SEQ ID NO: 1-3 or 5, wherein the difference consists of substitutinga glutamic acid or a aspartic acid with an arginine or a lysine allpositions 17, 19, and 21 of SEP ID NO: 1-3 or 5, or (ii) SEQ ID NO: 4,wherein the difference consists of substituting a glutamic acid or anaspartic acid with an arginine or a lysine at positions 18, 20, and 22of SEQ ID NO: 4, where the modified protein is permeable to a cell; b) apeptide linker region linked al either the amino or carboxyl end of themodified green fluorescent protein, the linker region susceptible todigestion by a specific protease; and c) a fluorescence modifier moietylinked to the end of the linker region, the fluorescence modifierchanging the fluorescence properties of the modified fluorescent proteinthat it is linked to, wherein the engineered protein, having the peptidelinker region and the fluorescent modifier moiety, is capable ofdetecting protease activity in a cells.
 14. The engineered protein asclaimed in claim 13, wherein the linker has the amino acid sequenceTSFNFPQITC (SEP ID NO: 13) and the protease is HIV-1.
 15. The engineeredprotein as claimed in claim 13, wherein the fluorescence modifyingmoiety is a tetramethyl rhodamine.
 16. An assay method for detectingprotease activity, the method comprising: exposing the engineeredprotein of claim 13 to at least one experimental cell; measuringfluorescence emission intensity of the cell to obtain a measuredfluorescence intensity; and comparing the measured fluorescenceintensity of die experimental cell with the fluorescence intensity of areference cell that has the engineered protein, but not a protease,wherein a difference in the fluorescence intensity between the two cellsis indicative of the presence of protease in the experimental cell.17.-18. (canceled)
 19. A kit for assaying cells for activity of aprotease, the kit comprising any one of the engineered green fluorescentproteins as claimed in claim
 13. 20. The kit as claimed in claim 19further comprising instructions for use.
 21. The method as claimed inclaim 16, wherein the at least one cell is from cells grown in cultureor a cell sample from a mammalian host.
 22. The method as claimed inclaim 16, wherein the protease is HIV-1.
 23. An assay method fordetecting protease activity, the method comprising: a) exposing anengineered protein to at least one experimental cell, wherein theprotein comprises i) a fluorescent protein differing in amino acidsequence from the reference protein of SEQ ID NO: 1-3 or 5, wherein thedifference consists of substituting a glutamic acid or a aspartic acidwith an arginine or a lysine at positions 17, 19, and 21 of SEQ ID NO:1-3 or 5, or SEQ ID NO: 4, wherein the difference consists ofsubstituting a glutamic acid or an aspartic acid with an arginine or alysine at positions 18, 20, and 22 of SEQ ID NO: 4, where the modifiedprotein is permeable to a cell; ii) a peptide linker region linked ateither the amino or carboxyl end of the modified fluorescent protein,the linker region susceptible to digestion by a specific protease; andiii) a fluorescence modifier moiety linked to the end of the linkerregion, the fluorescence modifier changing the fluorescencecharacteristics of the modified fluorescent protein that it is linkedto, wherein the engineered protein, having the peptide linker region andthe fluorescent modifier moiety, is capable of detecting proteaseactivity in the cell; b) measuring fluorescence emission intensity ofthe cell to obtain a measured fluorescence intensity; and c) comparingthe measured fluorescence intensity of the experimental cell with thefluorescence intensity of a reference cell that has the engineeredprotein, but not a protease, wherein a difference in the fluorescenceintensity between the two cells is indicative of the presence ofprotease in the experimental cell.